Project Summary Hedgehog (HH) proteins are a family of dual lipid-modified morphogens that mediate a complex signaling pathway known as the HH signaling pathway, which plays a key role in embryonic development and adult tissue homeostasis. The physiological significance of the HH pathway is further underscored by findings that its dysregulation has been implicated in multiple types of human cancers. The HH signal is transduced through its binding to the polytopic cell surface receptor Patched1 (PTCH1); this binding is enhanced by HH co-receptors. Binding of HH to PTCH1 relieves inhibition of the Frizzled-Class G-protein-coupled receptor Smoothened (SMO). Activated SMO subsequently triggers activation of the transcription factor glioma- associated oncogene (GLI), which enhances transcription of HH pathway target genes that drive cell proliferation. To elucidate molecular underpinnings of the HH pathway, we began by using cryogenic electron microscopy (cryo-EM) to determine the structure of human PTCH1 protein with native palmitoylated sonic hedgehog (SHH). These studies revealed the molecular basis through which the palmitate moiety of HH facilitates its binding to PTCH1 and how two PTCH1 molecules engage distinct sites on SHH, yielding a signaling-competent complex. It remains to be determined how HH co-receptors facilitate binding of HH to PTCH and how the signal is transduced from PTCH1 to SMO, triggering the HH signaling pathway. Our preliminary studies show that the oxysterol 24(S), 25-epoxycholesterol (24,25-EC), which we found associates with PTCH1, activates SMO and thereby permits recruitment of G proteins that initiate the HH signaling pathway. Building on our preliminary studies, we are now poised to 1) delineate mechanisms through which HH co-receptors augment HH?PTCH1 binding; 2) explore at the molecular level how 24(S),25- EC modulates SMO-Gi and SMO-Arrestin complexes; and 3) elucidate roles of SMO-associated proteins in regulation of the HH signaling pathway. Collectively, these studies will provide key insights into how HH is recognized at the cell membrane and modulates gene transcription through the action of a SMO-responsive G- protein. In addition, our studies will have significant clinical implications. SMO is the target of vismodegib, a widely prescribed drug for treatment of skin cancers that is also in the clinical trial stage for metastatic colorectal, advanced stomach, and pancreatic cancers. The current studies may reveal new therapeutic targets in the HH signaling pathway for treatment of these disorders.